Oliveira Natalia Kronbauer, Bhattacharya Somanon, Gambhir Rina, Joshi Manav, Fries Bettina C
Department of Microbiology and Immunology, Renaissance School of Medicine, Stony Brook University, Stony Brook, NY 11794, USA.
Division of Infectious Diseases, Department of Medicine, Stony Brook University, Stony Brook, NY 11794, USA.
J Fungi (Basel). 2022 Jun 28;8(7):677. doi: 10.3390/jof8070677.
causes meningoencephalitis in immunocompromised individuals, which is treated with fluconazole (FLC) monotherapy when resources are limited. This can lead to azole resistance, which can be mediated by overexpression of ABC transporters, a class of efflux pumps. ABC pump-mediated efflux of FLC is also augmented in 10-generation old cells. Here, we describe a new ABC transporter Afr3 (), which is overexpressed in cells of advanced generational age that accumulate during chronic infection. The Δ mutant strain showed higher FLC susceptibility by FLC E-Test strip testing and also by a killing test that measured survival after 3 h FLC exposure. Furthermore, Δ cells exhibited lower Rhodamine 6G efflux compared to the H99 wild-type cells. Afr3 was expressed in the ADΔ strain, which lacks several drug transporters, thus reducing background transport. The ADΔ + Afr3 strain demonstrated a higher efflux with both Rhodamine 6G and Nile red, and a higher FLC resistance. Afr3-GFP localized in the plasma membrane of the ADΔ + Afr3 strain, further highlighting its importance as an efflux pump. Characterization of the Δ mutant revealed unattenuated growth but a prolongation (29%) of the replicative life span. In addition, Δ exhibited decreased resistance to macrophage killing and attenuated virulence in the infection model. In summary, our data indicate that a novel ABC pump Afr3, which is upregulated in cells of advanced age, may contribute to their enhanced FLC tolerance, by promoting drug efflux. Lastly, its role in macrophage resistance may also contribute to the selection of older cells during chronic infection.
在免疫功能低下的个体中引发脑膜脑炎，当资源有限时，可用氟康唑（FLC）单一疗法进行治疗。这可能导致唑类耐药，其可由ABC转运蛋白（一类外排泵）的过表达介导。ABC泵介导的FLC外排在传代10次的细胞中也会增强。在此，我们描述了一种新的ABC转运蛋白Afr3（），其在慢性感染期间积累的高龄代细胞中过表达。通过FLC E-Test试纸条检测以及通过测量FLC暴露3小时后的存活率的杀伤试验，Δ突变株显示出更高的FLC敏感性。此外，与H99野生型细胞相比，Δ细胞表现出较低的罗丹明6G外排。Afr3在缺乏几种药物转运蛋白的ADΔ菌株中表达，从而减少背景转运。ADΔ + Afr3菌株对罗丹明6G和尼罗红均表现出更高的外排，以及更高的FLC耐药性。Afr3-GFP定位于ADΔ + Afr3菌株的质膜中，进一步突出了其作为外排泵的重要性。对Δ突变体的表征显示其生长未减弱，但复制寿命延长了29%。此外，在感染模型中，Δ对巨噬细胞杀伤的抗性降低且毒力减弱。总之，我们的数据表明，一种新的ABC泵Afr3在高龄细胞中上调，可能通过促进药物外排而有助于其增强的FLC耐受性。最后，其在巨噬细胞抗性中的作用也可能有助于在慢性感染期间选择较老的细胞。
